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Production and characterization of monoclonal antibodies to Xenopus Proteins

非洲爪蟾蛋白单克隆抗体的生产和表征

基本信息

批准号：
9208974
负责人：
DOMINIQUE R ALFANDARI
金额：
$ 29.52万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-07 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9208974
关键词：
Affinity Chromatography Amphibia Animals Antibodies Antibody Affinity Antigens Archives Biological Assay Biological Models Birds Cell Line Cell Surface Proteins Cell physiology Cell surface Cells Charge Chimeric Proteins Communities Competence Complex Data Development Developmental Process Disease Dorsal Embryo Epitopes Female Freezing Gene Silencing Goals HE protein Hybridomas Immunization Immunize Immunofluorescence Immunologic Immunoglobulin G Immunoprecipitation Individual Ion Exchange Libraries Ligands Mammalian Cell Mammals Masks Mass Spectrum Analysis Membrane Proteins Mesoderm Modeling Monoclonal Antibodies Mus Nature Neural Crest Cell Nuclear Paper Pattern Peptides Post-Translational Protein Processing Process Production Protein Family Proteins Proteomics Rana Receptor Signaling Recombinant Proteins Recombinants Research Personnel Resolution Resources Screening Result Serum Signaling Protein Spemann&apos s Organizer Spleen Testing Tissues Vertebral column Work Xenopus Xenopus Proteins Xenopus laevis antibody libraries base cell type chromatin immunoprecipitation cross reactivity design embryo membrane embryonic protein genome sequencing glycosylation in vivo Model liquid chromatography mass spectrometry meetings nuclear transfer polyclonal antibody protein complex relating to nervous system success tool transcription factor

项目摘要

PROJECT SUMMARY: The amphibian embryo and in particular the frog Xenopus laevis is an outstanding model used by embryologist and cell biologist worldwide. This model system has led the discovery of axis formation (antero/posterior and dorso/ventral), tissue induction and nuclear reprograming among others. Advances in genome sequencing, proteomics and gene silencing have rejuvenated this model. In its most recent White Paper, the Xenopus community has identified a lack of adequate resource of antibody to study Xenopus proteins and proposed the production of such resources a priority. We propose to produce at least a hundred fully characterized monoclonal antibodies to transcription factors, cell surface proteins including signaling receptors and ligands as well as tissue specific markers. To achieve this goal we will immunize mice with both selected recombinant proteins expressed in mammalian cells and complex protein extracts isolated from Xenopus embryos. The recombinant protein targets will be selected by a steering committee composed of Xenopus investigators based on the importance of the proteins in developmental and diseases processes and the absence of cross-reactive commercial antibodies. From these immunizations we will produce and archive more than 10,000 clonal IgG-expressing hyrbidomas that will be screened by immunofluor- escence, whole mount immunostaining and immunoprecipitation to identify monoclonal anti-bodies to native proteins. Final characterization of monoclonal antibodies produced against the complex embryonic extracts will be performed by mass spectrometry followed by immuno-fluorescence on the recombinant putative target(s). These monoclonal antibodies, together with the characterization data will be freely distributed to the community via the developmental hybridoma study bank and Xenbase.

项目概要：两栖动物胚胎，特别是青蛙非洲爪蟾是一个杰出的模型，胚胎学家和细胞生物学家。这个模型系统导致了轴形成的发现（前/后和背/腹）、组织诱导和核重编程等。基因组测序、蛋白质组学和基因沉默的进展使这一模型重新焕发了活力。在在其最新的白色文件中，非洲爪蟾社区已经确定缺乏足够的资源，抗体，研究爪蟾蛋白质，并提出生产此类资源的优先事项。我们我计划生产至少一百种完全表征的单克隆抗体，细胞表面蛋白，包括信号受体和配体以及组织特异性标记。为了实现这一目标，我们将用两种选定的重组蛋白免疫小鼠表达于哺乳动物细胞和从非洲爪蟾胚胎分离的复合蛋白提取物中。重组蛋白靶点将由非洲爪蟾组成的指导委员会选择研究人员基于蛋白质在发育和疾病过程中的重要性，缺乏交叉反应的商业抗体。从这些免疫中，我们将产生和存档超过10，000个克隆IgG表达的汗血瘤，这些汗血瘤将通过免疫荧光筛选，荧光、整体包埋免疫染色和免疫沉淀以鉴定单克隆抗体天然蛋白质。针对复合物产生的单克隆抗体的最终表征胚胎提取物将通过质谱法进行，然后在重组推定靶标。这些单克隆抗体，连同表征数据将通过开发杂交瘤研究库和Xenbase免费分发给社区。